Project Summary Humans are constantly exposed to a variety of chemicals via food, household products, medicines, and the environment. The kidney is particularly susceptible to chemical damage; drug-induced nephrotoxicity is a major health concern that contributes to 25% of all cases of severe acute kidney failure. Nephrotoxicity is also a significant problem in drug development; it is a major cause of attrition late in the process, accounting for 19% of failed Phase III clinical trials. The limitations of state-of-the-art animal models and in vitro tools for predicting human nephrotoxicity are well acknowledged, which has sparked the development of advanced 3D in vitro models of human tissues and organs, so called organ-on-chip systems (OOC) or microphysiological systems (MPS). While progress with human-based MPS has been rapid, a translational gap remains between MPS data and in vivo data. Therefore, comparing observations from animal-based MPS to in vivo animal data will inform our ability to translate human MPS findings to clinical medicine. This fast-track grant application proposes the development of kidney proximal tubule (KPT) MPS from two animal species that are frequently used in kidney toxicity screening: rat and dog. Once successfully developed, validated, and commercialized, the KPT-MPS will serve as an important new tool in chemical toxicity screening and drug development, allowing cross-referencing animal-based MPS data with in vivo animal data, human- based MPS data and clinical outcomes. It also has the potential to replace some animal testing, significantly reducing the use of live animals in preclinical testing. Phase I of the project is designed to establish robust KPT-MPS models from rat and dog. The project will leverage the commercially available Nortis MPS platform and pre-established protocols for creating human based KPT- MPS. Phase I/AIM 1 will demonstrate that proximal tubule cells from rat and dog form viable and structurally complete proximal tubules and that these tubules exhibit a stress response when exposed to compounds that are toxic to KPTs in vivo (Phase I/AIM 2). Feasibility requirements will be established in at least one of the two species before the project progresses into Phase II. Phase II will focus on optimizing the rat and or dog KPT- MPS prototypes and on developing assays for testing potentially nephrotoxic compounds (Phase II/AIM 1). For these efforts we will leverage a new microfluidic chip made of an injection-molded thermoplastic material that was developed for a human-based KPT-MPS. During Phase II/AIM 2, a panel of five compounds with published species-specific nephrotoxicity for rat, dog, and human will be tested in the KPT-MPS with the goal to establish correlation between KPT-MPS data and in vivo data in all three species. Phase II/AIM 3 is to demonstrate that rat/dog KPT-MPSs can be shipped to future customers as a plug & play pre-seeded product.